RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination

Nila Roy Choudhury; Gregory Heikel; Maryia Trubitsyna; Peter Kubik; Jakub Stanislaw Nowak; Shaun Webb; Sander Granneman; Christos Spanos; Juri Rappsilber; Alfredo Castello; Gracjan Michlewski

To detect endogenous RNA substrates of TRIM25, we performed UV crosslinking and immunoprecipitations in HeLa cells transiently expressing T7-tagged TRIM25, followed by high-throughput sequencing of TRIM25-associated RNAs (CLIP-seq). After UV crosslinking and immunoprecipitation, we observed a specific RNA-protein complex ( Figure S3a ). CLIPseq of T7-TRIM25ΔRBD resulted in a lack of detectable RNA-protein interactions (data not shown). The RNA from the T7-TRIM25/RNA complex was isolated using a CLIP-seq protocol and sequenced on a HiSeq platform. Data analysis and cluster identification using pyCRAC (Webb et al., 2014) revealed a significant correlation between three biological replicates with a correlation coefficient between clusters from 0.94 to 0.96 ( Figure S4 ). Five thousand five hundred and forty-nine clusters were common between all three experiments, and we identified 2611 distinct transcripts (Table S3) . Importantly, there was no correlation between transcript abundance, measured by a separate RNA-seq assay, and the cluster intensities, with a Pearson correlation of 0.1 ( Figure S5 ). This shows that TRIM25 does not simply bind the most abundant RNAs but has a substrate preference. Among the identified transcripts, mRNAs and lincRNAs were the most abundant (Fig. 4a ). GO-term annotation of TRIM25-bound transcripts revealed several nodes including RNA processing and translation, WD repeats, phosphorylation and ubiquitination ( Figure S6a ).